PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) infection kills more people a year than any other single infectious agent. Both the disease outcome and the pathology of TB are driven by the immune response mounted in the host, which is dictated by interactions between cells involved in innate and adaptive immunity. Because of the pivotal role of the immune response in TB, there is a growing interest in developing immunotherapies that harness the immune response to control the infection. In particular, there is interest in stimulating autophagy to control Mtb infection. The role of autophagy in controlling Mtb infection was based on the observation that Atg5fl/fl-LysM-cre mice lacking Atg5 in monocyte-derived cells and neutrophils (polymorphonuclear cells, PMN) succumb to Mtb within 30 days, an extremely severe phenotype similar to mice lacking IFN-? signaling. Atg5 is an essential autophagy protein and the dogma in the field was that Atg5 was required in macrophages to target Mtb for lysosomal degradation through macroautophagy. Contrary to expectation, we have demonstrated that Atg5 is required in myeloid cells to control Mtb infection due to an autophagy-independent function that regulates PMN-dominated inflammation. In addition, using mice that conditionally delete Atg5 only in PMN (Atg5fl/fl- MRP8-cre), we have shown that loss of Atg5 in PMNs can result in susceptibility to Mtb infection, revealing a PMN intrinsic role for Atg5 during Mtb infection. Therefore, it will be important to better understand the roles for autophagy proteins in host defense to understand the scope of effects that may occur while intervening with autophagic flux with host-directed therapies. Our dissection of Mtb pathogenesis in Atg5fl/fl-LysM-cre mice has revealed multiple stages where Atg5 functions in myeloid cells during infection. During Mtb infection, an early- infected Atg5-/- cell overproduces cytokines and chemokines that bring PMN into the lungs of Atg5fl/fl-LysM-cre mice in higher numbers than in control mice. The responding Atg5-/- PMN amplify the pro-inflammatory signals and a subset of Atg5-/- PMN are not cleared from the lung, which we predict occurs due to a defect in PMN apoptosis and/or efferocytosis of the apoptotic PMN. Efferocytosis of apoptotic Mtb-infected PMN is important for efficient antigen presentation. Indeed, the defect in clearing Atg5-/- PMN correlates with delayed trafficking of T cells to the lungs. Based on these preliminary data, I hypothesize that Atg5 plays roles in multiple myeloid cells during Mtb infection, including an autophagy-independent function in PMN, and together these functions of Atg5 lead to control of Mtb pathogenesis. To test this hypothesis, I will determine the mechanistic basis for how loss of Atg5 in myeloid cells leads to PMN accumulation and precludes a protective adaptive immune response to Mtb by pursuing the following aims: 1) Dissect the roles for Atg5 in regulating production of pro- inflammatory signals during infection, 2) Define the roles for Atg5 in regulating PMN accumulation and clearance during Mtb infection, 3) Determine how loss of Atg5 in myeloid cells affects adaptive immune responses to Mtb.